Human Growth Gene and Short Stature Gene Region

ABSTRACT

Subject of the present invention is an isolated human nucleic acid molecule encoding polypeptides containing a homeobox domain of sixty amino acids having the amino acid sequence of SEQ ID NO: 1 and having regulating activity on human growth. Three novel genes residing within the about 500 kb short stature critical region on the X and Y chromosome were identified. At least one of these genes is responsible for the short stature phenotype. The cDNA corresponding to this gene may be used in diagnostic tools, and to further characterize the molecular basis for the short stature-phenotype. In addition, the identification of the gene product of the gene provides new means and methods for the development of superior therapies for short stature.

This application is a divisional of U.S. Ser. No. 10/158,160 filed May31, 2002, which is a continuation of U.S. Ser. No. 09/147,699, filedJun. 24, 1999, now abandoned, which is a 371 of PCT/EP97/05355, filed onSep. 29, 1997, which claims the benefit of U.S. provisional application60/027,633, filed on Oct. 1, 1996.

The present invention relates to the isolation, identification andcharacterization of newly identified human genes responsible fordisorders relating to human growth, especially for short stature orTurner syndrome, as well as the diagnosis and therapy of such disorders.

The isolated genomic DNA or fragments thereof can be used forpharmaceutical purposes or as diagnostic tools or reagents foridentification or characterization of the genetic defect involved insuch disorders. Subject of the present invention are further humangrowth proteins (transcription factors A, B and C) which are expressedafter transcription of said DNA into RNA or mRNA and which can be usedin the therapeutic treatment of disorders related to mutations in saidgenes. The invention further relates to appropriate cDNA sequences whichcan be used for the preparation of recombinant proteins suitable for thetreatment of such disorders. Subject of the invention are furtherplasmid vectors for the expression of the DNA of these genes andappropriate cells containing such DNAs. It is a further subject of thepresent invention to provide means and methods for the genetic treatmentof such disorders in the area of molecular medicine using an expressionplasmid prepared by incorporating the DNA of this invention downstreamfrom an expression promotor which effects expression in a mammalian hostcell.

Growth is one of the fundamental aspects in the development of anorganism, regulated by a highly organised and complex system. Height isa multifactorial trait, influenced by both environmental and geneticfactors. Developmental malformations concerning body height are commonphenomena among humans of all races. With an incidence of 3 in 100,growth retardation resulting in short stature account for the largemajority of inborn deficiencies seen in humans.

With an incidence of 1:2500 life-born phenotypic females, Turnersyndrome is a common chromosomal disorder (Rosenfeld et al., 1996). Ithas been estimated that 1-2% of all human conceptions are 45,X and thatas many as 99% of such fetuses do not come to term (Hall and Gilchrist,1990; Robins, 1990). Significant clinical variability exists in thephenotype of persons with Turner syndrome (or Ullrich-Turner syndrome)(Ullrich, 1930; Turner, 1938). Short stature, however, is a consistentfinding and together with gonadal dysgenesis considered as the leadsymptoms of this disorder. Turner syndrome is a true multifactorialdisorder. Both the embryonic lethality, the short stature, gonadaldysgenesis and the characteristic somatic features are thought to be dueto monosomy of genes common to the X and Y chromosomes. The diploiddosis of those X-Y homologous genes are suggested to be requested fornormal human development. Turner genes (or anti-Turner genes) areexpected to be expressed in females from both the active and inactive Xchromosomes or Y chromosome to ensure correct dosage of gene product.Haploinsufficiency (deficiency due to only one active copy),consequently would be the suggested genetic mechanism underlying thedisease.

A variety of mechanisms underlying short stature have been elucidated sofar. Growth hormone and growth hormone receptor deficiencies as well asskeletal disorders have been described as causes for the short staturephenotype (Martial et al., 1979; Phillips et al., 1981; Leung et al.,1987; Goddard et al., 1995). Recently, mutations in three humanfibroblast growth factor receptor-encoding genes (FGFR 1-3) wereidentified as the cause of various skeletal disorders, including themost common form of dwarfism, achondroplasia (Shiang et al., 1994;Rousseau et al., 1994; Muenke and Schell, 1995). A well-known andfrequent (1:2500 females) chromosomal disorder, Turner Syndrome (45,X),is also consistently associated with short stature. Taken together,however, all these different known causes account for only a smallfraction of all short patients, leaving the vast majority of shortstature cases unexplained to date.

The sex chromosomes X and Y are believed to harbor genes influencingheight (Ogata and Matsuo, 1993). This could be deduced fromgenotype-phenotype correlations in patients with sex chromosomeabnormalities. Cytogenetic studies have provided evidence that terminaldeletions of the short arms of either the X or the Y chromosomeconsistently lead to short stature in the respective individuals(Zuffardi et al., 1982; Curry et al., 1984). More than 20 chromosomalrearrangements associated with terminal deletions of chromosome Xp andYp have been reported that localize the gene(s) responsible for shortstature to the pseudoautosomal region (PAR1) (Ballabio et al., 1989,Schaefer et al., 1993). This localisation has been narrowed down to themost distal 700 kb of DNA of the PAR1 region, with DXYS 15 as theflanking marker (Ogata et al., 1992; 1995).

Mammalian growth regulation is organized as a complex system. It isconceivable that multiple growth promoting genes (proteins) interactwith one another in a highly organized way. One of those genescontrolling height has tentatively been mapped to the pseudoautosomalregion PAR1 (Ballabio et al., 1989), a region known to be freelyexchanged between the X and Y chromosomes (for a review see Rappold,1993). The entire PAR1 region is approximately 2,700 kb.

The critical region for short stature has been defined with deletionpatients. Short stature is the consequence when an entire 700 kb regionis deleted or when a specific gene within this critical region ispresent in haploid state, is interrupted or mutated (as is the case withidiotypic short stature or Turner sydrome). The frequency of Turner'ssyndrome is 1 in 2500 females worldwide; the frequency of this kind ofidiopathic short stature can be estimated to be 1 in 4.000-5.000persons. Turner females and some short stature individuals usuallyreceive an unspecific treatment with growth hormone (GH) for many yearsto over a decade although it is well known that they have normal GHlevels and GH deficiency is not the problem. The treatment of suchpatients is very expensive (estimated costs approximately 30.000 USDp.a.). Therefore, the problem existed to provide a method and means fordistinguishing short stature patients on the one side who have a geneticdefect in the respective gene and on the other side patients who do nothave any genetic defect in this gene. Patients with a genetic defect inthe respective gene—either a complete gene deletion (as in Turnersyndrome) or a point mutation (as in idiopathic short stature)—should besusceptible for an alternative treatment without human GH, which now canbe devised.

Genotype/phenotype correlations have supported the existence of a growthgene in the proximal part of Yq and in the distal part of Yp. Shortstature is also consistently found in individuals with terminaldeletions of Xp. Recently, an extensive search for male and femalepatients with partial monosomies of the pseudoautosomal region has beenundertaken. On the basis of genotype-phenotype correlations, a minimalcommon region of deletion of 700 kb DNA adjacent to the telomere wasdetermined (Ogata et al., 1992; Ogata et al., 1995). The region ofinterest was shown to lie between genetic markers DXYS20 (3cosPP) andDXYS15 (113D) and all candidate genes for growth control from within thePAR1 region (e.g., the hemopoietic growth factor receptor a; CSF2RA)(Gough et al., 1990) were excluded based on their physical location(Rappold et al., 1992). That is, the genes were within the 700 kbdeletion region of the 2.700 kb PAR1 region.

Deletions of the pseudoautosomal region (PAR1) of the sex chromosomeswere recently discovered in individuals with short stature andsubsequently a minimal common deletion region of 700 kb within PAR1 wasdefined. Southern blot analysis on DNA of patients AK and SS usingdifferent pseudoautosomal markers has identified an Xp terminal deletionof about 700 kb distal to DXYS 15 (113D) (Ogata et al, 1992; Ogata etal, 1995).

The gene region corresponding to short stature has been identified as aregion of approximately 500 kb, preferably approximately 170 kb in thePAR1 region of the X and Y chromosomes. Three genes in this region havebeen identified as candidates for the short stature gene. These geneswere designated SHOX (also referred to as SHOX93 or HOX93), (SHOX=shortstature homeobox-containing gene), pET92 and SHOT (SHOX-like homeoboxgene on chromosome three). The gene SHOX which has two separate splicingsites resulting in two variations (SHOX a and b) is of particularimportance. In preliminary investigations, essential parts of thenucleotide sequence of the short stature gene could be analysed (SEQ IDNo. 8). Respective exons or parts thereof could be predicted andidentified (e.g. exon I [G310]; exon II [ET93]; exon IV [G108]; pET92).The obtained sequence information could then be used for designingappropriate primers or nucleotide probes which hybridize to parts of theSHOX gene or fragments thereof. By conventional methods, the SHOX genecan then be isolated. By further analysis of the DNA sequence of thegenes responsible for short stature, the nucleotide sequence of exonsI-V could be refined (v. FIG. 1-3). The gene SHOX contains a homeoboxsequence (SEQ ID NO: I) of approximately 180 bp (v. FIG. 2 and FIG. 3),starting from the nucleotide coding for amino acid position 117 (Q) tothe nucleotide coding for amino acid position 176 (E), i.e. from CAG(440) to GAG (619). The homeobox sequence is identified as thehomeobox-pET93 (SHOX) sequence and two point mutations have been foundin individuals with short stature in a German (A1) and a Japanesepatient by screening up to date 250 individuals with idiopahtic shortstature. Both point mutations were found at the identical position andleading to a protein truncation at amino acid position 195, suggestingthat there may exist a hot spot of mutation. Due to the fact that bothmutations found, which lead to a protein truncation, are at theidentical position, it is possible that a putative hot spot ofrecombination exisits with exon 4 (G108). Exon specific primers cantherefore be used as indicated below, e.g. GCA CAG CCA ACC ACC TAG (for)or TGG AAA GGC ATC ATC CGT AAG (rev).

The above-mentioned novel homeobox-containing gene, SHOX, which islocated within the 170 kb interval, is alternatively spliced generatingtwo proteins with diverse function. Mutation analysis and DNA sequencingwere used to demonstrate that short stature can be caused by mutationsin SHOX.

The identification and cloning of the short stature critical regionaccording to the present invention was performed as follows: Extensivephysical mapping studies on 15 individuals with partial monosomy in thepseudoautosomal region (PAR1) were performed. By correlating the heightof those individuals with their deletion breakpoints a short stature(SS) critical region of approximately 700 kb was defined. This regionwas subsequently cloned as an overlapping cosmid contig using yeastartificial chromosomes (YACs) from PAR 1 (Ried et al., 1996) and bycosmid walking. To search for candidate genes for SS within thisinterval, a variety of techniques were applied to an approximately 600kb region between the distal end of cosmid 56G10 and the proximal end of51D11. Using cDNA selection, exon trapping, and CpG island cloning, thetwo novel genes were identified.

The position of the short stature critical interval could be refined toa smaller interval of 170 kb of DNA by characterizing three furtherspecific individuals (GA, AT and RY), who were consistently short. Toprecisely localize the rearrangement breakpoints of those individuals,fluorescence in situ hybridization (FISH) on metaphase chromosomes wascarried out using cosmids from the contig. Patient GA, with a terminaldeletion and normal height, defined the distal boundary of the criticalregion (with the breakpoint on cosmid 110E3), and patient AT, with an Xchromosome inversion and normal height, the proximal boundary (with thebreakpoint on cosmid 34F5). The Y-chromosomal breakpoint of patient RY,with a terminal deletion and short stature, was also found to becontained on cosmid 34F5, suggesting that this region contains sequencespredisposing to chromosome rearrangements.

The entire region, bounded by the Xp/Yp telomere, has been cloned as aset of overlapping cosmids. Fluorescence in situ hybridization (FISH)with cosmids from this region was used to study six patients with Xchromosomal rearrangements, three with normal height and three withshort stature. Genotype-phenotype correlations narrowed down thecritical short stature interval to 270 kb of DNA or even less as 170 kb,containing the gene or genes with an important role in human growth. Aminimal tiling path of six to eight cosmids bridging this interval isnow available for interphase and metaphase FISH providing a valuabletool for diagnostic investigations on patients with idiopathic shortstature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gene map of the SHOX gene including five exons which areidentified as follows: exon I: G310, exon II: ET93, exon III: ET45, exonIV: G108 and exons Va and Vb, whereby exons Va and Vb result from twodifferent splicing sites of the SHOX gene. Exon II and III contain thehomeobox sequence of 180 nucleotides.

FIGS. 2 and 3 are the nucleotide and predicted amino acid sequences ofSHOXa and SHOXb:

-   -   SHOX a: The predicted start of translation begins at nucleotide        92 with the first in-frame stop codon (TGA) at nucleotides        968-970, yielding an open reading frame of 876 bp that encodes a        predicted protein of 292 amino acids (designated as        transcription factor A or SHOXa protein, respectively). An        in-frame, 5′stop codon at nucleotide 4, the start codon and the        predicted termination stop codon are in bold. The homeobox is        boxed (starting from amino acid position 117 (Q) to 176 (E),        i.e. CAG thru GAG in the nucleotide sequence). The locations of        introns are indicated with arrows. Two putative polyadenylation        signals in the 3′untranslated region are underlined.    -   SHOX b: An open reading frame of 876 bp exists from A in the        first methionin at nucleotide 92 to the in-frame stop codon at        nucleotide 767-769, yielding an open reading frame of 675 bp        that encodes a predicted protein of 225 amino acids        (transcription factor B or SHOXb protein, respectively). The        locations of introns are indicated with arrows. Exons I-IV are        identical with SHOXa, exon V is specific for SHOX b. A putative        polyadenylation signal in the 3′ untranslated region is        underlined.

FIG. 4 are the nucleotide (SEQ ID NO:43) and predicted amino acid (SEQID NO: 16) sequence of SHOT. The predicted start of translation beginsat nucleotide 43 with the first in-frame stop codon (TGA) at nucleotides613-615, yielding an open reading frame of 573 bp that encodes apredicted protein of 190 amino acids (designated as transcription factorC or SHOT protein, respectively). The homeobox is boxed (starting fromamino acid position 11 (Q) to 70 (E), i.e. CAG thru GAG in thenucleotide sequence). The locations of introns are indicated witharrows. Two putative polyadenylation signals in the 3′untranslatedregion are underlined

FIG. 5 gives the exon/intron organization of the human SHOX gene and therespective positions in the nucleotide sequence (Intron/Exon sequences(SEQ ID NOS 44-49, respectively in order of appearance) and Exon/Intronsequences (SEQ ID NOS 50-55, respectively in order of appearance)).

BRIEF DESCRIPTION OF THE SEQ ID:

SEQ ID NO.1: translated amino acid sequence of the homeobox domain (180bp)SEQ ID NO.2: exon II (ET93) of the SHOX geneSEQ ID NO. 3: exon I (G310) of the SHOX geneSEQ ID NO. 4: exon III (ET45) of the SHOX geneSEQ ID NO. 5: exon IV (G108) of the SHOX geneSEQ ID NO. 6: exon Va of the SHOX geneSEQ ID NO. 7: exon Vb of the SHOX geneSEQ ID NO. 8: preliminary nucleotide sequence of the SHOX geneSEQ ID NO.9: ET92 geneSEQ ID NO.10: SHOXa sequence (see also FIG. 2)SEQ ID NO.1: transcription factor A (see also FIG. 2)SEQ ID NO. 12: SHOXb sequence (see also FIG. 3)SEQ ID NO. 13: transcription factor B (see also FIG. 3)SEQ ID NO. 14: SHOX geneSEQ ID NO. 15: SHOT sequenceSEQ ID NO. 16: transcription factor C (see also FIG. 4)

Since the target gene leading to disorders in human growth (e.g. shortstature region) was unknown prior to the present invention, thebiological and clinical association of patients with this deletion couldgive insights to the function of this gene. In the present study,fluorescence in situ hybridization (FISH) was used to examine metaphaseand interphase lymphocyte nuclei of six patients. The aim was to testall cosmids of the overlapping set for their utility as FISH probes andto determine the breakpoint regions in all four cases, therebydetermining the minimal critical region for the short stature gene.

Duplication and deletion of genomic DNA can be technically assessed bycarefully controlled quantitative PCR or dose estimation on Southernblots or by using RFLPs. However, a particularly reliable method for theaccurate distinction between single and double dose of markers is FISH,the clinical application of is presently routine. Whereas in interphaseFISH, the pure absence or presence of a molecular marker can beevaluated, FISH on metaphase chromosomes may provide a semi-quantitativemeasurement of inter-cosmid deletions. The present inventor hasdetermined that deletions of about 10 kb (25% of signal reduction) canstill be detected. This is of importance, as practically all diseasegenes on the human X chromosome have been associated with smaller andlarger deletions in the range from a few kilobases to several megabasesof DNA (Nelson et al., 1995).

Subject of the present invention are therefore DNA sequences orfragments thereof which are part of the genes responsible for humangrowth (or for short stature, respectively, in case of genetic defectsin these genes). Three genes responsible for human growth wereidentified: SHOX, pET92 and SHOT. DNA sequences or fragments of thesegenes, as well as the respective full length DNA sequences of thesegenes can be transformed in an appropriate vector and transfected intocells. When such vectors are introduced into cells in an appropriate wayas they are present in healthy humans, it is devisable to treat diseasesinvolved with short stature, i.e. Turners syndrome, by modern means ofgene therapy. For example, short stature can be treated by removing therespective mutated growth genes responsible for short stature. It isalso possible to stimulate the respective genes which compensate theaction of the genes responsible for short stature, i.e. by inserting DNAsequences before, after or within the growth/short stature genes inorder to increase the expression of the healthy allels. By suchmodifications of the genes, the growth/short stature genes becomeactivated or silent, respectively. This can be accomplished by insertingDNA sequences at appropriate sites within or adjacent to the gene, sothat these inserted DNA sequences interfere with the growth/shortstature genes and thereby activate or prevent their transcription. It isalso devisable to insert a regulatory element (e.g. a promotor sequence)before said growth genes to stimulate the genes to become active. It isfurther devisable to stimulate the respective promotor sequence in orderto overexpress—in the case of Turner syndrome—the healthy functionalallele and to compensate for the missing allele. The modification ofgenes can be generally achieved by inserting exogenous DNA sequencesinto the growth gene/short stature gene via homologous recombination.

The DNA sequences according to the present invention can also be usedfor transformation of said sequences into animals, such as mammals, viaan appropriate vector system. These transgenic animals can then be usedfor in vivo investigations for screening or identifying pharamceuticalagents which are useful in the treatment of diseases involved with shortstature. If the animals positively respond to the administration of acandidate compound or agent, such agent or compound or derivativesthereof would be devisable as pharmaceutical agents. By appropriatemeans, the DNA sequences of the present invention can also be used ingenetic experiments aiming at finding methods in order to compensate forthe loss of genes responsible for short stature (knock-out animals).

In a further object of this invention, the DNA sequences can also beused to be transformed into cells. These cells can be used foridentifying pharmaceutical agents useful for the treatment of diseasesinvolved with short stature, or for screening of such compounds orlibrary of compounds. In an appropriate test system, variations in thephenotype or in the expression pattern of these cells can be determined,thereby allowing the identification of interesting candidate agents inthe development of pharmaceutical drugs.

The DNA sequences of the present invention can also be used for thedesign of appropriate primers which hybridize with segments of the shortstature genes or fragments thereof under stringent conditions.Appropriate primer sequences can be constructed which are useful in thediagnosis of people who have a genetic defect causing short stature. Inthis respect it is noteworthy that the two mutations found occur at theidentical position, suggesting that a mutational hot spot exists.

In general, DNA sequences according to the present invention areunderstood to embrace also such DNA sequences which are degenerate tothe specific sequences shown, based on the degeneracy of the geneticcode, or which hybridize under stringent conditions with thespecifically shown DNA sequences.

The present invention encompasses especially the following aspects:

-   -   a) An isolated human nucleic acid molecule encoding polypeptides        containing a homeobox domain of sixty amino acids having the        amino acid sequence of SEQ ID NO: 1 and having regulating        activity on human growth.    -   b) An isolated DNA molecule comprising the nucleotide sequence        essentially as indicated in FIG. 2, FIG. 3 or FIG. 4, and        especially as shown in SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID        NO: 15.    -   c) DNA molecules capable of hybridizing to the DNA molecules of        item b).    -   d) DNA molecules of item c) above which are capable of        hybridization with the DNA molecules of item 2. under a        temperature of 60-70° C. and in the presence of a standard        buffer solution.    -   e) DNA molecules comprising a nucleotide sequence having a        homology of seventy percent or higher with the nucleotide        sequence of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 15 and        encoding a polypeptide having regulating activity on human        growth.    -   f) Human growth proteins having the amino acid sequence of SEQ        ID NO: 11, 13 or 16 or a functional fragment thereof.    -   g) Antibodies obtained from immunization of animals with human        growth proteins of item f) or antigenic variants thereof.    -   h) Pharmaceutical compositions comprising human growth proteins        or functional fragments thereof for treating disorders caused by        genetic mutations of the human growth gene.    -   i) A method of screening for a substance effective for the        treatment of disorders mentioned above under item h) comprising        detecting messenger RNA hybridizing to any of the DNA molecules        decribed in a)-e) so as to measure any enhancement in the        expression levels of the DNA molecule in response to treatment        of the host cell with that substance.    -   j) An expression vector or plasmid containing any of the nucleic        acid molecules described in a)-e) above which enables the DNA        molecules to be expressed in mammalian cells.    -   k) A method for the determination of the gene or genes        responsible for short stature in a biological sample of body        tissues or body fluids.

In the method k) above, preferably nucleotide amplification techniques,e.g. PCR, are used for detecting specific nucleotide sequences known topersons skilled in the art, and described, for example, by Mullis et al.1986, Cold Spring Harbor Symposium Quant. Biol. 51, 263-273, and Saikiet al., 1988, Science 239, 487-491, which are incorporated herein byreference. The short stature nucleotide sequences to be determined aremainly those represented by sequences SEQ ID No. 2 to SEQ ID No. 7.

In principle, all oligonucleotide primers and probes for amplifying anddetecting a genetic defect responsible for deminished human growth in abiological sample are suitable for amplifying a target short statureassociated sequence. Especially, suitable exon specific primer pairsaccording to the invention are provided by table 1. Subsequently, asuitable detection, e.g. a radioactive or non-radioactive label iscarried out.

TABLE 1 Exon Sense primer Antisense primer Product (bp) Ta (° C.) 5′-I(G310) SP 1 ASP 1 194 58 3′-I (G310) SP 2 ASP 2 295 58 II (ET93) SP 3ASP 3 262 76/72/68 III (ET45) SP 4 ASP 4 120 65 IV (G108) SP 5 ASP 5 15462 Va (SHOXa) SP 6 ASP 6 265 61explanation of the abbreviations for the primers:

SP1 ATTTCCAATGGAAAGGCGTAAATAAC SP2 ACGGCTTTTGTATCCAAGTCTTTTG SP3GCCCTGTGCCCTCCGCTCCC SP4 GGCTCTTCACATCTCTCTCTGCTTC SP5CCACACTGACACCTGCTCCCTTTG S5P6 CCCGCAGGTCCAGGCTCAGCTG ASP1CGCCTCCGCCGTTACCGTCCTTG ASP2 CCCTGGAGCCGGCGCGCAAAG ASP3CCCCGCCCCCGCCCCCGG ASP4 CTTCAGGTCCCCCCAGTCCCG ASP5CTAGGGATCTTCAGAGGAAGAAAAAG ASP6 GCTGCGCGGCGGGTCAGAGCCCCAG

Also, a single stranded RNA can be used as target. Methods for reversedtranscribing RNA into cDNA are also well known and described in Sambrooket al., Molecular Cloning: A Laboratory Manual, New York, Cold SpringHarbor Laboratory 1989. Alternatively, preferred methods for reversedtranscription utilize thermostable DNA polymerases having RT activity.

Further, the technique described before can be used for selecting thoseperson from a group of persons being of short stature characterized by agenetic defect and which allows as a consequence a more specific medicaltreatment.

In another subject of the present invention, the transcription factorsA, B and C can be used as pharmaceutical agents. These transcriptionfactors initiate a still unknown cascade of biological effects on amolecular level involved with human growth. These proteins or functionalfragments thereof have a mitogenic effect on various cells. Especially,they have an osteogenic effect. They can be used in the treatment ofbone diseases, such as e.g. osteoporosis, and especially all thosediseases involved with disturbance in the bone calcium regulation.

As used herein, the term “isolated” refers to the original derivation ofthe DNA molecule by cloning. It is to be understood however, that thisterm is not intended to be so limiting and, in fact, the presentinvention relates to both naturally occurring and synthetically preparedseqences, as will be understood by the skilled person in the art.

The DNA molecules of this invention may be used in forms of gene therapyinvolving the use of an expression plasmid prepared by incorporating anappropriate DNA sequence of this invention downstream from an expressionpromotor that effects expression in a mammalian host cell. Suitable hostcells are procaryotic or eucaryotic cells. Procaryotic host cells are,for example, E. coli, Bacillus subtilis, and the like. By transfectinghost cells with replicons originating from species adaptable to thehost, that is, plasmid vectors containing replication starting point andregulator sequences, these host cells can be transfected with thedesired gene or cDNA. Such vectors are preferably those having asequence that provides the transfected cells with a property (phenotype)by which they can be selected. For example, for E. coli hosts the strainE. coli K12 is typically used, and for the vector either pBR322 or pUCplasmids can be generally employed. Examples for suitable promoters forE. coli hosts are trp promotor, lac promotor or Ipp promotor. Ifdesired, secretion of the expression product through the cell membranecan be effected by connecting a DNA sequence coding for a signal peptidesequence at the 5′ upstream side of the gene. Eucaryotic host cellsinclude cells derived from vertebrates or yeast etc.. As a vertebratehost cell, COS cells can be used (Cell, 1981, 23: 175-182), or CHOcells. Preferably, promotors can be used which are positioned 5′upstream of the gene to be expressed and having RNA splicing positions,polyadenylation and transcription termination seqences.

The transcription factors A, B and C of the present invention can beused to treat disorders caused by mutations in the human growth genesand can be used as growth promoting agents. Due to the polymorphismknown in the case of eukaryotic genes, one or more amino acids may besubstituted. Also, one or more amino acids in the polypeptides can bedeleted or inserted at one or more sites in the amino acid sequence ofthe polypeptides of SEQ ID NO: 11, 13 or 16. Such polypeptides aregenerally referred to equivalent polypeptides as long as the underlyingbiological acitivity of the unmodified polypeptide remains essentiallyunchanged.

The present invention is illustrated by the following examples.

EXAMPLE 1 Patients

All six patients studied had de novo sex chromosome aberrations.

CC is a girl with a karyotype 45,X/46,X psu dic (X)(Xqter→Xp22.3::Xp22.3→Xqter). At the last examination at 6½ years ofage, her height was 114 cm (25-50 the % percentile). Her mother's heightwas 155 cm, the father was not available for analysis. For details, seeHenke et al., 1991.

GA is a girl with a karyotype 46,X der X (3pter→3p23::Xp22.3→Xqter). Atthe last examination at 17 years, normal stature (159 cm) was observed.Her mother's height is 160 cm and her father's height 182 cm. Fordetails, see Kulharya et al, 1995.

SS is a girl with a karyotype 46,X rea (X) (Xqter→Xq26::Xp22.3→Xq26:).At 11 years her height remained below the 3rd percentile growth curvefor Japanese girls; her predicted adult height (148.5 cm) was below hertarget height (163 cm) and target range (155 to 191 em). For details,see Ogata et alt, 1992.

AK is a girl with a karyotype 46,X rea (X)(Xqter→Xp22.3::Xp22.3→Xp21.3:). At 13 years her height remained belowthe 2nd percentile growth curve for Japanese girls; her predicted adultheight (142.8 cm) was below her target height (155.5 cm) and targetrange (147.5-163.5 em). For details, see Ogata et alt, 1995.

RY: the karyotype of the ring Y patient is 46,X,r(Y)/46,Xdicr(Y)/45,X[95:3:2], as examined on 100 lymphocytes; at 16 years of agehis final height was 148; the heights of his three brothers are all inthe normal range with 170 cm (16 years, brother 1), 164 cm (14 years,brother 2) and 128 cm (9 years, brother 3), respectively. Growthretardation of this patient is so severe that it would also becompatible with an additional deletion of the GCY locus on Yq.

AT: boy with ataxia and inv(X); normal height of 116 cm at age 7,parents' heights are 156 cm and 190 cm, respectively.

Patients for Mutation Analysis:

250 individuals with idiopathic short stature were tested for mutationsin SHOXa. The patients were selected on the following criteria: heightfor chronological age was below the 3rd centile of national heightstandards, minus 2 standard deviations (SDS); no causative disease wasknown, in particular: normal weight (length) for gestational age, normalbody proportions, no chronic organic disorder, normal food intake, nopsychiatric disorder, no skeletal dysplasia disorder, no thyroid orgrowth hormone deficiency.

Family A:

Cases 1 and 2 are short statured children of a German non-consanguineousfamily. The boy (case 1) was born at the 38th week of gestation bycesarian section. Birth weight was 2660 g, birth length 47 cm. Hedeveloped normally except for subnormal growth. On examination at theage of 6.4 years, he was proportionate small (106.8 cm, −2.6 SDS) andobese (22.7 kg), but otherwise normal. His bone age was not retarded (6yrs) and bone dysplasia was excluded by X-ray analysis. IGF-I andIGFBP-3 levels as well as thyroid parameters in serum rendered GH orthyroid hormone deficiency unlikely. The girl (case 2) was born at termby cesarian section. Birth weight was 2920 g, birth length 47 cm. Herdevelopmental milestones were normal, but by the age of 12 months poorgrowth was apparent (length: 67 cm, −3.0 SDS). At 4 years she was 89.6cm of height (−3.6 SDS). No dysmorphic features or dysproportions wereapparent. She was not obese (13 kg). Her bone age was 3.5 years and bonedysplasia was excluded. Hormone parameters were normal. It isinteresting to note that both the girl and the boy grow on the 50percentile growth curve for females with Turner syndrome. The mother isthe smallest of the family and has a mild rhizomelic dysproportion(142.3 cm, −3.8 SDS). One of her two sisters (150 cm, −2.5 SDS) and thematernal grandmother (153 cm, −2.0 SDS) are all short without anydysproportion. One sister has normal stature (167 cm, +0.4 SDS). Thefather's height is 166 cm (−1.8 SDS) and the maternal grandfather'height is 165 cm (−1.9 SDS). The other patient was of Japanese originand showed the identical mutation.

EXAMPLE 2 Identification of the Short Stature Gene

A. In situ Hybridizationa) Florescence in situ Hybridization (FISH)

Florescence in situ hybridization (FISH) using cosmids residing in theXp/Yp pseudoautosomal region (PAR1) was carried out. FISH studies usingcosmids 64/75cos (LLNLc 110H032), E22cos (2e2), F1/14cos (110A7),M1/70cos (110E3), P99F2cos (43C11), P99cos (LLNLc110P2410), B6cosb(1CRFc104H0425), F20cos (34F5), F21cos (ICRFc104G0411), F3cos2 (9E3),F3cos1 (11E6), P117cos (29B11), P6cos1 (ICRFc104P0117), P6cos2(LLNLc110E0625) and E4cos (15G7) was carried out according to publishedmethods (Lichter and Cremer, 1992). In short, one microgram of therespective cosmid clone was labeled with biotin and hybridized to humanmetaphase chromosomes under conditions that suppress signals fromrepetitive DNA sequences. Detection of the hybridization signal was viaFITC-conjugated avidin. Images of FITC were taken by using a cooledcharge coupled device camera system (Photometrics, Tucson, Ariz.).

b) Physical Mapping

Cosmids were derived from Lawrence Livermore National Laboratory X- andY-chromosome libraries and the Imperial Cancer Research Fund London (nowMax Planck Institute for Molecular Genetics Berlin) X chromosomelibrary. Using cosmids distal to DXYS15, namely E4cos, P6cos2, P6cos1,P1117cos and F3cos1 one can determine that two copies are still presentof E4cos, P6cos2, P6cos1 and one copy of P117cos and F3cos1. Breakpointsof both patients AK and SS map on cosmid P6cos1, with a maximum physicaldistance of 10 kb from each other. It was concluded that the abnormal Xchromosomes of AK and SS have deleted about 630 kb of DNA. Furthercosmids were derived from the ICRF X chromosome specific cosmid library(ICRFc104), the Lawrence Livermore X chromosome specific cosmid library(LLNLc110) and the Y chromosome specific library (LLCO3′M′), as well asfrom a self-made cosmid library covering the entire genome. Cosmids wereidentified by hybridisation with all known probes mapping to this regionand by using entire YACs as probes. To verify overlaps, end probes fromseveral cosmids were used in cases in which overlaps could not be provenusing known probes.

c) Southern Blot Hybridisation

Southern blot analysis using different pseudoautosomal markers hasprovided evidence that the breakpoint on the X chromosome of patient CCresides between DXYS20 (3cosPP) and DXYS60 (U7A) (Henke et al, 1991). Inorder to confirm this finding and to refine the breakpoint location,cosmids 64/75cos, E22cos, F1/14cos, M1/70cos, F2cos, P99F2cos and P99coswere used as FISH probes. The breakpoint location on the abnormal X ofpatient CC between cosmids 64/75cos (one copy) and F1/14cos (two copies)on the E22PAC could be determined. Patient CC with normal statureconsequently has lost approximately 260-290 kb of DNA.

Southern blot hybridisations were carried out at high stringencyconditions in Church buffer (0.5 M NaPi pH 7.2, 7% SDS, 1 mM EDTA) at65° C. and washed in 40 mM NaPi, 1% SDS at 65° C.

d) FISH Analysis

Biotinylated cosmid DNA (insert size 32-45 kb) or cosmid fragments(10-16 kb) were hybridised to metaphase chromosomes from stimulatedlymphocytes of patients under conditions as described previously(Lichter and Cremer, 1992). The hybridised probe was detected viaavidin-conjugated FITC.

e) PCR Amplification

All PCRs were performed in 50 μl volumes containing 100 pg-200 ngtemplate, 20 pmol of each primer, 200 μM dNTP's (Pharmacia), 1.5 mMMgCl₂, 75 mMTris/HCl pH9, 20 mM (NH₄)₂SO₄ 0.01% (w/v) Tween20 and 2 U ofGoldstar DNA Polymerase (Eurogentec). Thermal cycling was carried out ina Thermocycler GeneE (Techne).

f) Exon Amplification

Four cosmid pools consisting of each four to five clones from the cosmidcontigs were used for exon amplification experiments. The cosmids ineach cosmid pool were partially digested with Sau3A. Gel purifiedfractions in the size range of 4-10 kb were cloned in the BamHI digestedpSPL3B vector (Burn et al, 1995) and used for the exon amplificationexperiments as previously described (Church et al., 1994).

g) Genomic Sequencing

Sonificated fragments of the two cosmids LLOYNCO3′M′15D10 andLLOYNCO3′M′34F5 were subcloned separately into M13mp18 vectors. Fromeach cosmid library at least 1000 plaques were picked, M13 DNA preparedand sequenced using dye-terminators, ThermoSequenase (Amersham) anduniversal M13-primer (MWG-BioTech). The gels were run on ABI-377sequencers and data were assembled and edited with the GAP4 program(Staden).

Of all six patients, GA had the least well characterized chromosomalbreakpoint. The most distal markers previously tested for their presenceor absence on the X were DXS1060 and DXS996, which map approximately 6Mb from the telomere (Nelson et al., 1995). Several cosmids containingdifferent gene sequences from within PAR1 (MIC2, ANT3, CSF2RA, and XE7)were tested and all were present on the translocation chromosome.Cosmids from within the short stature critical region e.g., chromosome,thereby placing the translocation breakpoint on cosmid M1/70cos. Aquantitative comparison of the signal intensities of M1/70cos betweenthe normal and the rearranged X indicates that approximately 70% of thiscosmid is deleted.

TABLE 2 Table 2: This table summarizes the FISH data for the 16 cosmidstested on four patients. CC GA AK SS 64/75cos − − E22cos − − F1/14cos +− M1/70cos + (+) F2cos + + P99F2cos + + P99cos + + B6cos + F20cos F21cosF3cos2 F3cos1 − − P117cos − − P6cos1 + + P6cos2 + + E4cos + + [−] onecopy; indicates that the respective cosmid was deleted on the rearrangedX, but present on the normal X chromosome [+] two copies; indicates thatthe respective cosmid is present on the rearranged and on the normal Xchromosome [(+)] breakpoint region; indicates that the breakpoint occurswithin the cosmid as shown by FISH

In summary, the molecular analysis on six patients with X chromosomalrearrangements using florescence-labeled cosmid probes and in situhybridization indicates that the short stature critical region can benarrowed down to a 270 kb interval, bounded by the breakpoint of patientGA from its centromere distal side and by patients AK and SS on itscentromere proximal side.

Genotype-phenotype correlations may be informative and have been chosento delineate the short stature critical interval on the human X and Ychromosome. In the present study FISH analysis was used to studymetaphase spreads and interphase nuclei of lymphocytes from patientscarrying deletions and translocations on the X chromosome andbreakpoints within Xp22.3. These breakpoints appear to be clustered intwo of the four patients (AK and SS) presumably due to the presence ofsequences predisposing to chromosome rearrangements. One additionalpatient Ring Y has been found with an interruption in the 270 kbcritical region, thereby reducing the critical interval to a 170 kbregion.

By correlating the height of all six individuals with their deletionbreakpoint, an interval of 170 kb was mapped to within thepseudoautosomal region, presence or absence of which has a significanteffect on stature. This interval is bounded by the X chromosomalbreakpoint of patient GA at 340 kb from the telomere (Xptel) distallyand by the breakpoints of patients AT and RY at 510/520 kb Xptelproximally. This assignment constitutes a considerable reduction of thecritical interval to almost one fourth of its previous size (Ogata etal., 1992; Ogata et al., 1995). A small set of six to eight cosmids arenow available for FISH experiments to test for the prevalence andsignificance of this genomic locus on a large series of patients withidiopathic short stature.

B. Identification of the Candidate Short Stature Gene

To search for transcription units within the smallest 170 kb criticalregion, exon trapping and cDNA selection on six cosmids (110E3, F2cos,43C11, P2410, 15D10, 34F5) was carried out. Three different positiveclones (ET93, ET45 and G108) were isolated by exon trapping, all ofwhich mapped back to cosmid 34F5. Previous studies using cDNA selectionprotocols and an excess of 25 different cDNA libraries had provenunsuccessful, suggesting that genes in this interval are expressed atvery low abundancy.

To find out whether any gene in this interval was missed, the nucleotidesequence of about 140 kb from this region of the PAR1 was determined,using the random M13 method and dye terminator chemistry. The cosmidsfor sequence analysis were chosen to minimally overlap with each otherand to collectively span the critical interval. DNA sequence analysisand subsequent protein prediction by the “X Grail” program, version 1.3cas well as by the exon-trapping program FEXHB were carried out andconfirmed all 3 previously cloned exons. No protein-coding genes otherthan the previously isolated one could be detected.

C. Isolation of the Short Stature Candidate Gene SHOX

Assuming that all three exon clones ET93, ET45 and G108 are part of thesame gene, they were used collectively as probes to screen 14 differentcDNA libraries from 12 different fetal (lung, liver, brain 1 and 2) andadult tissues (ovary, placenta 1 and 2, fibroblast, skeletal muscle,bone marrow, brain, brain stem, hypothalamus, pituitary). Not a singleclone among approximately 14 million plated clones was detected. Toisolate the full-length transcript, 3′ and 5′RACE were carried out. For3′RACE, primers from exon G108 were used on RNA from placenta, skeletalmuscle and bone marrow fibroblasts, tissues where G108 was shown to beexpressed in. Two different 3′RACE clones of 1173 and 652 bp werederived from all three tissues, suggesting that two different 3′exons aand b exist. The two different forms were termed SHOXa and SHOXb.

To increase chances to isolate the complete 5′portion of a gene known tobe expressed at low abundancy, a Hela cell line was treated withretinoic acid and phorbol ester PMA. RNA from such an induced cell lineand RNA from placenta and skeletal muscle were used for the constructionof a ‘Marathon cDNA library’. Identical 5′RACE cDNA clones were isolatedfrom all three tissues.

Experimental Procedure:

RT-PCR and cDNA Library Construction

Human polyA⁺RNA of heart, pancreas, placenta, skeletal muscle, fetalkidney and liver was purchased from Clontech. Total RNA was isolatedfrom a bone marrow fibroblast cell line with TRIZOL reagent (Gibco-BRL)as described by the manufacturer. First strand cDNA synthesis wasperformed with the Superscript first strand cDNA synthesis kit(Gibco-BRL) starting with 100 ng polyA⁺RNA or 10 μg total RNA usingoligo(dt)-adapter primer (GGCCACGCGTCGACTAGTAC[dT]₂₀N. After firststrand cDNA synthesis the reaction mix was diluted 1/10. For further PCRexperiments 5 μl of this dilutions were used.

A ‘Marathon CDNA library’ was constructed from skeletal muscle andplacenta polyA⁺ RNA with the marathon cDNA amplification kit (Clontech)as described by the manufacturer.

Fetal brain (catalog # HL5015b), fetal lung (HL3022a), ovary (HL1098a),pituitary gland (HL1097v) and hypothalamus (HL1172b) cDNA libraries werepurchased from Clontech. Brain, kidney, liver and lung cDNA librarieswere part of the quick screen human cDNA library panel (Clontech). Fetalmuscle cDNA library was obtained from the UK Human Genome MappingProject Resource Center.

D. Sequence Analysis and Structure of SHOX Gene

A consensus sequence of SHOXa and SHOXb (1349 and 1870 bp) was assembledby analysis of sequences from the 5′ and 3′RACE derived clones. A singleopen reading frame of 1870 bp (SHOXa) and 1349 bp (SHOXb) wasidentified, resulting in two proteins of 292 (SHOXa) and 225 amino acids(SHOXb). Both transcripts a and b share a common 5′end, but have adifferent last 3′exon, a finding suggestive of the use of alternativesplicing signals. A complete alignment between the two cDNAs and thesequenced genomic DNA from cosmids LL0YNCO3″M″15D10 and LL0YNC3″M″34F5was achieved, allowing establishment of the exon-intron structure (FIG.4). The gene is composed of 6 exons ranging in size from 58 bp (exonIII) to 1146 bp (exon Va). Exon I contains a CpG-island, the start codonand the 5′ region. A stop codon as well as the 3′-noncoding region islocated in each of the alternatively spliced exons Va and Vb.

EXAMPLE 3

Two cDNAs have been identified which map to the 160 kb region identifiedas critical for short stature. These cDNAs correspond to the genes SHOXand pET92. The cDNAs were identified by the hybridization of subclonesof the cosmids to cDNA libraries.

Employing the set of cosmid clones with complete coverage of thecritical region has now provided the genetic material to identify thecausative gene. Positional cloning projects aimed at the isolation ofthe genes from this region are done by exon trapping and cDNA selectiontechniques. By virtue of their location within the pseudoautosomalregion, these genes can be assumed to escape X-inactivation and to exerta dosage effect.

The cloning of the gene leading to short stature when absent (haploid)or deficient, represents a further step forward in diagnostic accuracy,providing the basis for mutational analysis within the gene by e.g.single strand conformation polymorphism (SSCP). In addition, cloning ofthis gene and its subsequent biochemical characterization has opened theway to a deeper understanding of biological processes involved in growthcontrol.

The DNA sequences of the present invention provide a first moleculartest to identify individuals with a specific genetic disorder within thecomplex heterogeneous group of patients with idiopathic short stature.

EXAMPLE 4 Expression Pattern of SHOXa and SHOXb

Northern blot analysis using single exons as hybridisation probesreveiled a different expression profile for every exon, stronglysuggesting that the bands of different size and intensities representcross-hybridisation products to other G,C rich gene sequences. Toachieve a more realistic expression profile of both genes SHOXa and b,RT-PCR experiments on RNA from different tissues were carried out.Whereas expression of SHOXa was observed in skeletal muscle, placenta,pancreas, heart and bone marrow fibroblasts, expression of SHOXb wasrestricted to fetal kidney, skeletal muscle and bone marrow fibroblasts,with the far highest expression in bone marrow fibroblasts.

The expression of SHOXa in several cDNA libraries made of fetal brain,lung and muscle, of adult brain, lung and pituitary and of SHOXb in noneof the tested libraries gives additional evidence that one spliced form(SHOXa) is more broadly expressed and the other (SHOXb) expressed in apredominantly tissue-specific manner.

To assess the transcriptional activity of SHOXa and SHOXb on the X and Ychromosome we used RT-PCR of RNA extracted from various cell linescontaining the active X, the inactive X or the Y chromosome as the onlyhuman chromosomes. All cell lines revealed an amplification product ofthe expected length of 119 bp (SHOXa) and 541 bp (SHOXb), providingclear evidence that both SHOXa and b escape X-inactivation.

SHOXa and SHOXb encode novel homeodomain proteins. SHOX is highlyconserved across species from mammalian to fish and flies. The very 5′end and the very 3′ end—besides the homeodomain—are likely conservedregions between man and mouse, indicating a functional significance.Differences in those amino acid regions have not been allowed toaccumulate during evolution between man and mouse.

Experimental Procedures: a) 5′ and 3′RACE

To clone the 5′ end of the SHOXa and b transcripts, 5′RACE was performedusing the constructed ‘Marathon cDNA libraries’. The followingoligonucleotide primers were used: SHOX B rev, GAAAGGCATCCGTAAGGCTCCC(position 697-718, reverse strand [r]) and the adaptor primer AP1. PCRwas carried out using touchdown parameters: 94° C. for 2 min, 94° C. for30 sec, 70° C. for 30 sec, 72° C. for 2 min for 5 cycles. 94° C. for 30sec, 66° C. for 30 sec, 72° C. for 2 min for 5 cycles. 94° C. for 30sec, 62° C. for 30 sec, 72° C. for 2 min for 25 cycles. A second roundof amplification was performed using 1/100 of the PCR product and thefollowing nested oligonucleotide primers: SHOX A rev,GACGCCTTTATGCATCTGATTCTC (position 617-640 r) and the adaptor primerAP2. PCR was carried out for 35 cycles with an annealing temperature of60° C.

To clone the 3′ end of the SHOXa and b transcripts, 3′RACE was performedas previously described (Frohman et al., 1988) using oligo(dT)adaptorprimed first strand cDNA. The following oligonucleotide primers wereused: SHOX A for, GAATCAGATGCATAAAGGCGTC (position 619-640) and theoligo(dT)adaptor. PCR was carried out using following parameters: 94° C.for 2 min, 94° C. for 30 sec, 62° C. for 30 sec, 72° C. for 2 min for 35cycles. A second round of amplification was performed using 1/100 of thePCR product and the following nested oligonucleotide primers: SHOX Bfor, GGGAGCCTTACGGATGCCTTTC (position 697-718) and the oligo(dT)adaptor.PCR was carried out for 35 cycles with annealing temperature of 62° C.

To validate the sequences of SHOXa and SHOXb transcripts, PCR wasperformed with a 5′ oligonucleotide primer and a 3′ oligonucleotideprimer. For SHOXa the following primers were used: G310 for,AGCCCCGGCTGCTCGCCAGC (position 59-78) and SHOX D rev,CTGCGCGGCGGGTCAGAGCCCCAG (position 959-982 r). For SHOXb the followingprimers were used: G310 for, AGCCCCGGCTGCTCGCCAGC and SHOX2A rev,GCCTCAGCAGCAAAGCAAGATCCC (position 1215-1238 r). Both PCRs were carriedout using touchdown parameters: 94° C. for 2 min, 94° C. for 30 sec, 70°C. for 30 sec, 72° C. for 2 min for 5 cycles. 94° C. for 30 sec, 68° C.for 30 sec, 72° C. for 2 min for 5 cycles. 94° C. for 30 sec, 65° C. for30 sec, 72° C. for 2 min for 35 cycles. Products were gel-purified andcloned for sequencing analysis.

b) SSCP Analysis

SSCP analysis was performed on genomic amplified DNA from patientsaccording to a previously described method (Orita et al., 1989). One tofive μl of the PCR products were mixed with 5 μl of denaturationsolution containing 95% Formamid and 10 mM EDTA pH8 and denaturated at95° C. for 10 min. Samples were immediately chilled on ice and loaded ona 10% Polyacryamidgel (Acrylamide:Bisacryamide=37.5:1 and 29:1;Multislotgel, TGGE base, Qiagen) containing 2% glycerol and 1×TBE. Gelswere run at 15° C. with 500V for 3 to 5 hours and silver stained asdescribed in TGGE handbook (Qiagen, 1993).

c) Cloning and Sequencing of PCR Products

PCR products were cloned into pMOSBlue using the pMOSBlueT- Vector Kitfrom Amersham. Overnight cultures of single colonies were lysed in 100μl H₂O by boiling for 10 min. The lysates were used as templates forPCRs with specific primers for the cloned PCR product. SSCP of PCRproducts allowed the identification of clones containing differentalleles. The clones were sequenced with CY5 labelled vector primers Uniand T7 by the cycle sequencing method described by the manufacturer(ThermoSequenase Kit (Amersham)) on an ALF express automated sequencer(Pharmacia).

d) PCR Screening of cDNA Libraries

To detect expression of SHOXa and b, a PCR screening of several cDNAlibraries and first strand cDNAs was carried out with SHOXa and bspecific primers. For the cDNA libraries a DNA equivalent of 5×10⁸ pfuwas used. For SHOXa, primers SHOX E rev, GCTGAGCCTGGACCTGTTGGAAAGG(position 713-737 r) and SHOX a for were used. For SHOXb, the followingprimers were used: SHOX B for and SHOX2A rev. Both PCRs were carried outusing touchdown parameters: 94° C. for 2 min; 94° C. for 30 sec, 68° C.for 30 sec, 72° C. for 40 sec for 5 cycles. 94° C. for 30 sec, 65° C.for 30 sec, 72° C. for 40 sec for 5 cycles. 94° C. for 30 sec, 62° C.for 30 sec, 72° C. for 40 sec for 35 cycles.

e) PCR Screening of cDNA Libraries

To detect expression of SHOXa and b, a PCR screening of several cDNAlibraries and first strand cDNAs was carried out with SHOXa and bspecific primers. For the cDNA libraries a DNA equivalent of 5×10⁸ pfuwas used. For SHOXa, primers SHOX E rev, GCTGAGCCTGGACCTGTTGGAAAGG(position 713-737 r) and SHOX a for were used. For SHOXb, the followingprimers were used: SHOX B for and SHOX2A rev. Both PCRs were carried outusing touchdown parameters: 94° C. for 2 min; 94° C. for 30 sec, 68° C.for 30 sec, 72° C. for 40 sec for 5 cycles. 94° C. for 30 sec, 65° C.for 30 sec, 72° C. for 40 sec for 5 cycles. 94° C. for 30 sec, 62° C.for 30 sec, 72° C. for 40 sec for 35 cycles.

EXAMPLE 5 Expression Pattern of OG12, the Putative Mouse Homolog of BothSHOX and SHOT

In situ hybridisation on mouse embryos ranging from day 5 p.c. and day18.5 p.c., as well as on fetal and newborn animals was carried out toestablish the expression pattern. Expression was seen in the developinglimb buds, in the mesoderm of nasal processes which contribute to theformation of the nose and palate, in the eyelid, in the aorta, in thedeveloping female gonads, in the developing spinal cord (restricted todifferentiating motor neurons) and brain. Based on this expressionpattern and on the mapping position of its human homolog SHOT, SHOTrepresents a likely candidate for the Cornelia de Lange syndrome whichincludes short stature.

EXAMPLE 6 Isolation of a Novel SHOX-Like Homeobox Gene on ChromosomeThree, SHOT, Being Related to Human Growth/Short Stature

A new gene called SHOT (for SHOX-homolog on chromosome three) wasisolated in human, sharing the most homology with the murine OG12 geneand the human SHOX gene. The human SHOT gene and the murine OG12 genesare highly homologous, with 99% identity at the protein level. Althoughnot yet proven, due to the striking homology between SHOT and SHOX (identity within the homeodomain only), it is likely that SHOT is also agene likely involved in short stature or human growth.

SHOT was isolated using primers from two new human ESTs (HS 1224703 andHS 126759) from the EMBL database, to amplify a reverse-transcribed RNAfrom a bone marrow fibroblast line (Rao et al, 1997). The 5′ and 3′ endsof SHOT were generated by RACE-PCR from a bone marrow fibroblast librarythat was constructed according to Rao et al., 1997. SHOT was mapped byFISH analysis to chromosome 3q25/q26 and the murine homolog to thesyntenic region on mouse chromosome 3. Based on the expression patternof OG12, its mouse homolog, SHOT represents a candidate for the CorneliaLange syndrome (which shows short stature and other features, includingcraniofacial abnormalities) mapped to this chromosomal interval on3q25/26.

EXAMPLE 7 Searching for Mutations in Patients with Idiopathic ShortStature

The DNA sequences of the present invention are used in PCR, LCR, andother known technologies to determine if such individuals with shortstature have small deletions or point mutations in the short staturegene.

A total of initially 91 (in total 250 individuals) unrelated male andfemale patients with idiopathic short stature (idiopathic short staturehas an estimated incidence of 2-2.5% in the general population) weretested for small rearrangements or point mutations in the SHOXa gene.Six sets of PCR primers were designed not only to amplify single exonsbut also sequences flanking the exon and a small part of the 5′UTR. Forthe largest exon, exon one, two additional internal-exon primers weregenerated. Primers used for PCR are shown in table 2.

Single strand conformation polymorphism (SSCP) of all amplified exonsranging from 120 to 295 bp in size was carried out. Band mobility shiftswere identified in only 2 individuals with short stature (Y91 and A1).Fragments that gave altered SSCP patterns (unique SSCP conformers) werecloned and sequenced. To avoid PCR and sequencing artifacts, sequencingwas performed on two strands using two independent PCR reactions. Themutation in patient Y91 resides 28bp 5′ of the start codon in the 5′UTRand involves a cytidine-to-guanine substitution. To find out if thismutation represents a rare polymorphism or is responsible for thephenotype by regulating gene expression e.g. though a weaker binding oftranslation initiation factors, his parents and a sister were tested. Asboth the sister and father with normal height also show the same SSCPvariant (data not shown), this base substitution represents a rarepolymorphism unrelated to the phenotype.

Cloning and sequencing of a unique SSCP conformer for patient A1revealed a cytidine-to-thymidine base transition (nucleotide 674) whichintroduces a termination codon at amino-acid position 195 of thepredicted 225 and 292 amino-acid sequences, respectively. To determinewhether this nonsense mutation is genetically associated with the shortstature in the family, pedigree analysis was carried out. It was foundthat all six short individuals (defined as height below 2 standarddeviations) showed an aberrant SSCP shift and the cytidine-to-thymidinetransition. Neither the father, nor one aunt and maternal grandfatherwith normal height showed this mutation, indicating that the grandmotherhas transferred the mutated allele onto two of her daughters and her twograndchildren. Thus, there is concordance between the presence of themutant allele and the short stature phenotype in this family.

The identical situation as indicated above was found in another shortstature patient of Japanese origin.

EXAMPLE 8

The DNA sequences of the present invention are used to characterize thefunction of the gene or genes. The DNA sequences can be used as searchqueries for data base searching of nucleic acid or amino acid databasesto identify related genes or gene products. The partial amino acidsequence of SHOX93 has been used as a search query of amino aciddatabases. The search showed very high homology to many known homeoboxproteins. The cDNA sequences of the present invention can be used torecombinantly produce the peptide. Various expression systems known tothose skilled in the art can be used for recombinant protein production.

By conventional peptide synthesis (protein synthesis according to theMerrifield method), a peptide having the sequence CSKSFDQKSKDGNGG (SEQID NO: 42) was synthesized and polyclonal antibodies were derived inboth rabbits and chicken according to standard protocols.

REFERENCES

The following references are herein incorporated by reference.

Ashworth A, Rastan S, Lovell-Badge R, Kay G (1991): X-chromosomeinactivation may explain the difference in viability of X0 humans andmice. Nature 351: 406-408.

Ballabio A, Bardoni A, Carrozzo R, Andria G, Bick D, Campbell L, HamelB, Ferguson-Smith MA, Gimelli G, Fraccaro M, Maraschio P, Zuffardi O,Guilo S, Camerino G (1989): Contiguous gene syndromes due to deletionsin the distal short arm of the human X chromosome. Proc Natl Acad SciUSA 86:10001-10005.

Blagowidow N, Page D C, Huff D, Mennuti M T (1989): Ullrich-Tumersyndrome in an XY female fetus with deletion of the sex-determiningportion of the Y chromosome. Am. J. med. Genet. 34:159-162.

Cantrell M A, Bicknell J N, Pagon R A et al. (1989): Molecular analysisof 46,XY females and regional assignment of a new Y-chromosome-specificprobe. Hum. Genet. 83: 88-92.

Connor J M, Loughlin S A R (1989): Molecular genetics of Turner'ssyndrome. Acta Pediatr. Scand. (Suppl.) 356: 77-80.

Disteche C M, Casanova M, Saal H, Friedmen C, Sybert V, Graham J,Thuline H, Page D C, Fellous M (1986): Small deletions of the short armof the Y-chromosome in 46,XY females. Proc Natl Acad Sci USA83:7841-7844.

Ferguson-Smith M A (1965): Karyotype-phenotype correlations in gonadaldysgenesis and their bearing on the pathogenesis of malformations. J.med. Genet. 2: 142-155.

Ferrari D, Kosher R A, Dealy C N (1994): Limb mesenchymal cellsinhibited from undergoing cartilage differentiation by a tumor promotingphorbol ester maintain expression of the homeobox-containing gene MSX1and fail to exhibit gap junctional communication. Biochemical andBiophysical Research Communications. 205(1): 429-434.

Fischer M, Bur-Romero P, Brown L G et al. (1990): Homologous ribosomalprotein genes in the human X- and Y-chromosomes escape fromX-inactivation and possible implementation for Turner syndrome. Cell 63:1205-1218.

Freund C, Horsford D J, McInnes R R (1996): Transcription factor genesand the developing eye: a genetic perspective. Hum Mol Genet 5:1471-1488.

Gehring W J, Qian Y Q, Billeter M, Furukubo-Tokunaga K, Schier A F,Resendez-Perez D, Affolter M, Otting G, Wüthrich K (1994):Homeodomain-DNA recognition. Cell 78: 211-223.

Gough N M, Gearing D P, Nicola N A, Baker E, Pritchard M, Callen D F,Sutherland G R (1990). Localization of the human GM-CSF receptor gene tothe X-Y pseudoautosomal region. Nature 345: 734736

Grumbach M M, Conte F A (1992): Disorders of sexual differentiation. In:Williams textbook of endocrinology, 8th edn., edited by Wilson J D,Foster D W, pp. 853-952, Philadelphia, W B Saunders.

Hall J G, Gilchrist D M (1990): Turner syndrome and its variants.Pedriatr. Clin. North Am. 37: 1421-1436.

Henke A, Wapenaar M, van Ommen G-J, Maraschio P, Camerino O, Rappold G A(1991): Deletions within the pseudoautosomal region help map three newmarkers and indicate a possible role of this region in linear growth. AmJ Hum Genet 49:811-819.

Hernandez D, Fisher E M C (1996): Down syndrome genetics: unravelling amultifactorial disorder. Hum Mol Genet 5:1411-1416.

Kenyon C (1994): If birds can fly, why can't we? Homeotic genes andevolution. Cell 78: 175-180.

Krumlauf R (1994): Hox genes in vertebrate development. Cell 78:191-201.

Kulharya A S, Roop H, Kukolich M K, Nachtman R G, Belmont J W,Garcia-Heras J (1995): Mild phenotypic effects of a de novo deletionXpter→Xp22.3 and duplication 3pter→3p23. Am J Med Genet 56:16-21.

Lawrence P A, Morata G (1994): Homeobox genes: their function inDrosophila segmentation and pattern formation. Cell 78: 181-189.

Lehrach H, Drmnac R, Hoheisel J D, Larin Z, Lemon G, Monaco A P, NizeticD, et a,. Hybridization finger printing in genome mapping andsequencing. In Davies K E, Tilghman S, Eds. Genome Analysis 1990: 39-81Cold Spring Harbor, N.Y.

Levilliers J, Quack B, Weissenbach J, Petit C (1989): Exchange ofterminal portions of X- and Y-chromosomal short arms in human XYfemales. Proc Natl Acad Sci USA 86:2296-2300.

Lichter P, Cremer T, Human Cytogenetics: A practical Approach, IRL Press1992, Oxford, New York, Tokyo

Lippe B M (1991): Turner Syndrome. Endocrinol Metab Clin North Am 20:121-152. Magenis R E, Tochen M L Holahan K P, Carey T, Allen L, Brown MG (1984): Turner syndrome resulting from partial deletion ofY-chromosome short arm: localization of male determinants. J Pediatr105: 916-919.

Nelson D L, Ballabio A, Cremers F, Monaco A P, Schlessinger D(1995).—Report of the sixth international workshop on the X chromosomemapping. Cytogenet. Cell Genet. 71: 308-342

Ogata T, Goodfellow P, Petit C, Aya M, Matsuo N (1992): Short stature ina girl with a terminal Xp deletion distal to DXYS15: localization of agrowth gene(s) in the pseudoautosomal region. J Med Genet 29:455-459.

Ogata T, Tyler-Smith C, Purvis-Smith S, Turner G (1993): Chromosomallocalisation of a gene(s) for Turner stigmata on Yp. J. Med. Genet. 30:918-922.

Ogata T, Yoshizawa. A, Muroya K, Matsuo N, Fukushima Y, Rappold GA,Yokoya S (1995): Short stature in a girl with partial monosomy of thepseudoautosomal region distal to DXYS15: further evidence for theassignment of the critical region for a pseudoautosomal growth gene(s).J Med Genet 32:831-834.

Ogata T, Matsuo N (1995): Turner syndrome and female sex chromosomeaberrations: deduction of the principle factors involved in thedevelopment of clinical features. Hum. Genet. 95: 607-629.

Orita M, Suzuki Y, Sekiya T and Hayashi K (1989): Rapid and sensitivedetection of point mutations and polymorphisms using the polymerasechain reaction. Genomics 5:874-879.

Pohlschmidt M, Rappold G A, Krause M, Ahlert D, Hosenfeld D, WeissenbachJ, Gal A (1991): Ring Y chromosome: Molecular characterization by DNAprobes. Cytogenet Cell Genet 56:65-68.

Qiagen (1993) TGGE Handbook, Diagen GmbH, TGMA 4112 3/93.

Rao E, Weiss B, Mertz A et al. (1995): Construction of a cosmid contigspanning the short stature candidate region in the pseudoautosomalregion PAR 1. in: Turner syndrome in a life span perspective: Researchand clinical aspects. Proceedings of the 4th International Symposium onTurner Syndrome, Gothenburg, Sweden, 18-21 May, 1995., edited byAlbertsson-Wikland K, Ranke M B, pp. 19-24, Elsevier.

Rao E, Weiss B, Fukami M, Rump A, Niesler B, Mertz A, Muroya K, BinderG, Kirsch S, Winkelmann M, Nordsiek G, Heinrich U, Breuning M H, Ranke MB, Rosenthal A, Ogata T, Rappold G A (1997): Pseudoautosomal deletionsencompassing a novel homeobox gene cause growth failure in idiopathicshort stature and Turner syndrome. Nature Genet 15:54-62

Rappold G A (1993): The pseudoautosomal region of the human sexchromosomes. Hum Genet 92:315-324.

Rappold G A, Willson T A, Henke A, Gough N M (1992): Arrangement andlocalization of the human GM-CSF receptor α chain gene CSF2RA within theX-Y pseudoautosomal region. Genomics 14:455-461.

Ried K, Mertz A, Nagaraja R, Trusnich M, Riley J, Anand R, Page D,Lehrach H., Elliso J, Rappold G A (1995): Characterization of a yeastartificial chromosome contig spanning the pseudoautosomal region.Genomics 29:787-792.

Robinson A (1990): Demography and prevalence of Turner syndrome. In:Turner Syndrome., edited by Rosenfeld R G, Grumbach M M, pp. 93-100, NewYork, Marcel Dekker.

Rosenfeld R G (1992): Turner syndrome: a guide for physicians. Secondedition. The Turner's Syndrome Society.

Rosenfeld R G, Tesch L-G, Rodriguez-Rigau L J, McCauley E,Albertsson-Wikland K, Asch R, Cara J, Conte F, Hall J G, Lippe B, NagelT C, Neely E K, Page D C, Ranke M, Saenger P, Watkins J M, Wilson D M(1994): Recommendations for diagnosis,treatment, and management ofindividuals with Turner syndrome. The Endocrinologist 4(5): 351-358.

Rovescalli A C, Asoh S, Nirenberg M (1996): Cloning and characterizationof four murine homeobox genes. Proc Natl Acad Sci USA 93:10691-10696.

Schaefer L, Ferrero G B, Grillo A, Bassi M T, Roth E J, Wapenaar M C,van Ommen G-J B, Mohandas T K, Rocchi M, Zoghbi H Y, Ballabio A (1993):A high resolution deletion map of human chromosome Xp22. Nature genetics4: 272-279.

Shalet S M (1993): Leukemia in children treated with growth hormone.Journal of Pediatric Endocrinology 6: 109-11

Vimpani G V, Vimpani A F, Lidgard G P, Cameron E H D, Farquhar J W(1977) Prevalence of severe growth hormone deficiency. Br Med J. 2:427-430

Zinn A R, Page D C, Fisher E M C (1993): Turner syndrome: the case ofthe missing sex chromosome. TIG 9 (3): 90-93.

1. A method for the treatment of short stature in patients identified ashaving a genetic: defect in the human growth gene SHOX, comprising a)identifying a genetic defect in a human subject suspected of having agenetic mutation in the SHOX gene having the nucleotide sequenceaccording to SEQ. ID NO. 14, and b) administering to said patient atherapeutically active amount of human growth hormone.
 2. The methodaccording to claim 1, wherein said genetic defect is identified byobtaining a biological sample molecule to be examined, and amplifyingsaid biological sample molecule in the presence of two nucleotide probescompletely or in part complementary to any of the DNA sequences of SEQ.ID. NO: 2 to SEQ ID NO.
 7. 3. The method according to claim 1, whereinthe genetic mutation is caused by a hot spot of mutation in the nucleicacid sequence encoding a protein truncation at amino acid position 195in the SHOX gene.
 4. The method according to claim 1, wherein saidpatient is not suffering from Turner's Syndrome.
 5. A method for thetreatment of short stature in patients identified as having a geneticdefect in the human growth gene SHOX, said SHOX gene having thenucleotide sequence according to SEQ. ID NO. 14, comprising a)identifying a genetic defect in a human subject suspected of having agenetic mutation in the SHOX gene, comprising i) obtaining a biologicalsample containing a polynucleotide from a human subject, ii) amplifyingthe polynucleotide of i) in the presence of a primer, wherein saidprimer is an exon flanking primer or a primer to an exon nucleotidesequence of the SHOX gene according to SEQ ID NO: 14 and wherein theoligonucleotide primer has a length of 18-26 nucleotides and theoligonucleotide sequence of said primer is identical to a partialsequence of an exon nucleotide sequence of the SHOX gene according toSEQ ID NO: 14, and iii) sequencing any amplification product of thepolynucleotide of i) to determine the presence of a genetic mutation inthe SHOX gene of said human subject, and b) administering to said humansubject a therapeutically active amount of human growth hormone.
 6. Themethod according to claim 5, wherein the exon nucleotide sequence instep a) ii) is a polynucleotide sequence selected from the groupconsisting of SHOX ET93 (SEQ ID NO: 2), SHOX G310 (SEQ ID NO: 3), SHOXET45 (SEQ ID NO: 4), SHOX G108 (SEQ ID NO: 5), SHOX Va (SEQ ID NO: 6)and SHOX Vb (SEQ ID NO: 7).